Influenza a virus remains a health menace and the rapidly evolving nature of this segmented RNA virus leads to the emergence of new, unseen subtypes, which have potential to cause a viral pandemic. Influenza nucleoprotein (NP) is a multifunctional viral protein whose role in regulating viral RNA synthesis has not been fully elucidated. The aim of the proposed research is to fill the gap in knowledge regarding the role of NP in viral RNA synthesis and processing in vivo. NP is highly homologous among influenza A isolates and thus antiviral therapies targeting NP are expected to have efficacy against multiple influenza A subtypes. The aim of this research strategy is to discover biochemical interactions of NP required for influenza RNA synthesis in order to define molecular mechanisms underlying influenza gene expression and identify novel antiviral targets with the long term goal to spur development of innovative antiviral therapies. The objective is to characterize two NP mutants which display RNA synthesis defects and were designed to disrupt interaction with either the viral RNA dependent RNA polymerase (RdRP) or host RNA processing factor UAP56. The central hypothesis is that these interactions are important for viral RNA synthesis in vivo. The rationale for the proposed research is that characterization of these NP mutants will delineate viral-viral interactions suitable as antiviral targets. This central hypothesis will be tested by pursuing the following two specific aims: 1) Characterize an influenza NP mutant targeted for disruption of interaction with the viral RdRP; and 2) Define the role of N-terminal NP interactions required for viral RNA synthesis. To analyze these NP mutants for RNA synthesis and processing activities, an innovative transfection system will be employed to examine expression from vRNA(-) and/or cRNA(+) templates. This innovative strategy is beneficial to alleviate the public fear associated with generation of recombinant mutant influenza viruses in the laboratory, but more important this strategy will allow identification of NP mutants which might prove lethal to the virus and thus would not be recovered as a recombinant mutant virus. Aim 1 focuses on the viral-viral interaction between NP and RdRP. Aim 2 targeted a viral-host interaction but as shown in the preliminary results, this NP mutant is rescued by viral NS, and will thus likely define important viral-viral interactions. The strong preliminary data demonstrate feasibility of the proposed experiments to characterize these NP mutants. Tools to perform immuno purification and gel shift assays are already on hand and established as feasible in the applicant's laboratory. The expected outcomes will have a positive impact by identifying and characterizing important viral-viral interactions which may serve as novel antiviral targets. The work will make a significant contribution by defining amino acids of NP involved in essential viral-viral interactions and lay the foundation for development of antiviral therapies targeting these NP interactions and effective against multiple influenza A subtypes to control influenza related disease.